Differential effects of short-chain fatty acids on proliferation and production of pro- and anti-inflammatory cytokines by cultured lymphocytes
Introduction
Acetic, propionic, and butyric acids are the predominant forms of volatile short-chain fatty acids (SCFA) in the gastrointestinal tract (Bergman, 1990). They are produced by fermentation of water-soluble fiber by anaerobic bacteria normally resident in the large bowel Cummings and Branch, 1986, Bergman, 1990. Although the relative proportions of the SCFA produced in the large bowel vary according to the type of fiber in the diet Cummings and Branch, 1986, Bergman, 1990, Clausen, 1991, Folino et al., 1995, typical concentrations present are 10 mM (acetate), 1.5 mM (butyrate), and 2 mM (propionate) (Bergman, 1990). Studies indicate that the nature of fiber in the diet can affect the composition, metabolism and function of cells of the immune system Calder et al., 1996, Cavaglieri Felippe et al., 1997, Lim et al., 1997, Cavaglieri et al., 2000. These effects of fiber may be due to the SCFA produced, since SCFA have been demonstrated to influence immune cell responses. For example, acetate, butyrate and propionate have been shown to inhibit T lymphocyte proliferation in vitro Franklin et al., 1991, Curi et al., 1993, Wajner et al., 1999.
Inflammatory bowel diseases (IBD) are characterised by chronic inflammation of the gut mucosa Sartor, 1995, Brandtzaeg et al., 1997, Dionne et al., 1999. Abberant T lymphocyte responses underlie this inflammation Fuss et al., 1996, Fiocchi, 1998, Dionne et al., 1999. In particular, a dysregulation of T helper cell phenotype in favour of T helper-type 1 (Th1) cells appears to underlie Crohn's disease, one form of IBD Fuss et al., 1996, Fiocchi, 1998, Dionne et al., 1999. Th1 cells are characterised by the production of the cytokines interleukin (IL)-2 and interferon (IFN)-γ (Mossman and Sad, 1996), which drive the inflammatory process. In contrast, Th2 cells are characterised by production of IL-4 which antagonises the production of Th1 cytokines (Mossman and Sad, 1996). IL-10 is produced by regulatory T lymphocytes (Mossman and Sad, 1996) and by monocytes and macrophages. IL-10 inhibits the production of Th1 cytokines (Mossman and Sad, 1996) and the production of inflammatory cytokines, such as tumor necrosis factor-α, by monocytes and macrophages (Ralph et al., 1992). Pro-inflammatory cytokines produced by Th1 cells and by monocytes and macrophages are responsible for some of the tissue damage seen in IBD Sartor, 1995, Brandtzaeg et al., 1997, Dionne et al., 1997, Dionne et al., 1999. IL-10 administration ameliorates gut inflammation in animal models and IL-10 knockout mice spontaneously develop IBD (Hagenbaugh et al., 1997). Consumption of fiber has been reported to lower risk of gut inflammation (Burkitt, 1984). This may be due to SCFA production. Indeed, butyrate enemas have been found to be as effective as drug therapy in some patients with IBD (Senagore et al., 1992). The efficacy of fiber and of butyrate may result from an alteration in T cell phenotype, with a shift away from the Th1 towards a more anti-inflammatory phenotype. However, the effect of SCFA on T cell phenotypes has been little studied. Therefore, the aim of this study was to identify the effects of the three common SCFA on the production of T cell-derived cytokines by cultured gut-associated lymphocytes.
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Materials
Concanavalin A (Con A), sodium acetate, sodium propionate, sodium butyrate, RPMI culture medium, fetal calf serum (FCS), glutamine and antibiotics were purchased from Sigma Chemical Co., Poole, Dorset, UK. [6-3H] Thymidine was purchased from Amersham Internacional, Amersham, Bucks., UK. Cytoscreen™ enzyme-linked immunosorbent assay (ELISA) kits for cytokine measurements were obtained from BioSource International, Camarillo, CA, USA.
Lymphocyte preparation culture and proliferation assay
Male Wistar rats (300–400 g) were sacrificed by an overdose of
Effect of SCFA on T lymphocyte proliferation
Thymidine incorporation in the absence of Con A stimulation was 267 ± 53 cpm/well. Con A stimulation markedly increased thymidine incorporation (Table 1). This was not significantly affected by the inclusion of acetate (10 mM) or propionate (2 mM) in the culture medium. However, butyrate (1.5 mM) significantly decreased thymidine incorporation (by more than 99%) (Table 1). Separate experiments investigating the effect of different concentrations of butyrate showed that its inhibitory effects
Discussion
Lymphocytes prepared from the mesenteric lymph nodes of healthy rats were predisposed to exhibit a Th1 phenotype: when stimulated with the T cell mitogen Con A they produced much higher concentrations of the Th1-type cytokines IL-2 and IFN-γ than of the anti-inflammatory cytokine IL-10. Furthermore, the Th2-type cytokine IL-4 was not produced under the cell culture conditions used. Including SCFA in the culture medium resulted in a modification of the lymphocyte response to Con A, in that the
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